Flaviviruses need and manipulate host cell pathways at every step of their infectious cycle. Since many host cell pathways are controlled by protein kinases, this class of host proteins is intimately involved in modulating flaviviral replication both negatively, ie. by controlling innate immune responses, as well as positively, by inducing a virus-supportive environment. The goal of this sub-project is to identify those kinase-controlled host cell pathways that support the flavivirus life cycle. Our preliminary data show that broad-spectrum protein tyrosine kinase inhibitors prevent flaviviral entry whereas MAP-kinase inhibitors interfere with replication. Moreover, we previously demonstrated that Src-kinase inhibitors block flaviviral egress due to a requirement of the c-yes kinase for this step. To identify both specific inhibitors and specific kinases controlling each step of the viral life cycle we will use a chemical genomics approach. We will employ cellbased, high content screening of kinase-directed small molecule libraries to identify kinase inhibitors interfering with different steps of the Dengue-virus (DENV) life cycle. In secondary screens we will select for compounds with a high selectivity index, broad cell type specificity and activity against other flaviviruses (WNV, JEV, YFV). Active compounds will likely inhibit several related kinases thus minimizing the problem of redundancy of signaling pathways and tissue specific expression. The kinases targeted by a given compound will be identified by combining computational, functional genomics and biochemical approaches. Based on these results we will generate models of kinase networks controlling flavivirus infection and examine thise models experimentally by phosphoproteomics and by targeted gene knockdown. Finally, we will examine the therapeutic potential of kinase-targeted antivirally active compounds by examining selected compounds in vivo using a murine model West-Nile Virus infection. The efficacy, toxicity and bio-availability of kinase-directed compounds will be compared with the in vivo activity of TYT-1 analogs (sultam thioureas) generated in sub-project 3 of this program. At the end of these comprehensive studies, we anticipate to have gained further insights into the interaction of flaviviruses with their host cell, identified novel targets for antviral drugs discovery, and characterized novel small molecule inhibitors that are active against flaviviruses in vitro and in vivo.